CN107106734A - The artificial cornea of three dimensional biological printing - Google Patents
The artificial cornea of three dimensional biological printing Download PDFInfo
- Publication number
- CN107106734A CN107106734A CN201580062303.9A CN201580062303A CN107106734A CN 107106734 A CN107106734 A CN 107106734A CN 201580062303 A CN201580062303 A CN 201580062303A CN 107106734 A CN107106734 A CN 107106734A
- Authority
- CN
- China
- Prior art keywords
- layers
- cec
- cepc
- hydrogel
- hypothallus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3886—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types
- A61L27/3891—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells comprising two or more cell types as distinct cell layers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3808—Endothelial cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3813—Epithelial cells, e.g. keratinocytes, urothelial cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
- A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
- A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
- A61L27/48—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix with macromolecular fillers
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M33/00—Means for introduction, transport, positioning, extraction, harvesting, peeling or sampling of biological material in or from the apparatus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0618—Cells of the nervous system
- C12N5/0621—Eye cells, e.g. cornea, iris pigmented cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/12—Nanosized materials, e.g. nanofibres, nanoparticles, nanowires, nanotubes; Nanostructured surfaces
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/16—Materials or treatment for tissue regeneration for reconstruction of eye parts, e.g. intraocular lens, cornea
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/30—Synthetic polymers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Abstract
By individually cultivating stroma cell living, the endothelial cell (CEC) of work and corneal epithelial cell (CEpC) living, and the single hypothallus of 3D biometric prints, CEC layers and CEpC layers carry out manufacture of intraocular cornea so that cell to be encapsulated into single hydrogel nanometer grid.The first side for being attached to the hypothallus by the CEC layers and the second side for being attached to the hypothallus by the CEpC layers, to limit artificial cornea.
Description
Related application
The rights and interests of the priority for the U.S. Provisional Application No. 62/054,924 submitted for 24th this application claims September in 2014,
This application is combined herein by quoting with its full text.
Invention field
The present invention relates to the 3D biometric prints of artificial organ, and relate more specifically to the people that produces using 3D biometric prints
Work cornea.
Background of invention
One or more layers disease or injury of cornea can cause blindness, generally be treated by corneal transplantation.
Annual about 40,000 patients in the U.S. receive corneal graft.It is most in these people to receive the replacement from non-human donor
Cornea.Although operation has high success rate, the supply of donor tissue is limited, and waiting list may be very long.In hair
It is national in exhibition, obtain donor tissue even more difficult.And, although non-human donor's transplanting is that the standard of corneal blindness is controlled
Treat, but wherein intrinsic complication and limitation promoted synthesis cornea substitute development.Existing synthesis cornea can
To be divided into：1) completely synthetic prosthese (such as corneal prostheses (keratoprostheses)) and 2) allow what host tissue regenerated
Hydrogel.
Corneal prostheses (keratoprostheses) or Kpro are most well-known artificial corneas, perform the dioptric work(of cornea
Energy.Although Kpro has existed for many years in a variety of forms, the transparency, biomethanics of the manufacture with non-human donor's cornea
Matrix equivalent with the synthesis of power of regeneration is still a difficult challenge.Moreover, the application of corneal prostheses is by complexity
Implant procedure and main postoperative complication (including infection, calcification, prosthese film (retroprosthetic membrane)
Formed and glaucoma) obstruction.In some cases, due to its infection tendency, patient must take antibiotic throughout one's life.Therefore,
In refusal naive donors tissue repeatedly or it is not eligible for carrying out the patient of such transfer operation originally, artificial cornea is used only as most
Means afterwards.
The engineering cornea of Second Type is the acellular implant based on synthesis hydrogel, and it is designed to restore host
Cell is with the growth epithelial layer on implant surface and recovers function.Many in these hydrogel implants, which is similar to, unit
Knit and the high elastic modulus with desired optical property.However, in most cases, being mechanically fixed or biological fixation
The integration for being the support and host tissue of problematic-implantation is extremely time-consuming process.This slow time course due to
Limited cytothesis activity in older and/or major injuries patients and be further exacerbated by.In addition, it was reported that these water-settings
Some in glue implant are partly biodegradable after being chronically implanted, and cause the transparency to be lost and graft failure.Processing is without thin
The trial of some problems of born of the same parents' implant, which is included in hydrogel matrix, mixes glucosaminoglycan, and these glucosaminoglycans are considered as
It is required for cell adherence and regulation degradability.
One of change application of bio-nanotechnology is that reconstruction and regeneration for human tissue and organ create revolutionary
Method.This promise is the great ability provided based on nanometer technology under Biological background：Controlled on nanoscale thin
The unique pattern of born of the same parents' machine.Due to the modularization framework of their special surfaces characteristic, subcellular fraction length dimension and accurate pointing,
Nanostructured and its incorporation in organizational project construct provide new example for regenerative medicine.3D biometric prints-it makes
3D knots are manufactured by addition manufacturing process as module is built with biomaterial, cell, protein and other biological compound
Structure-offer can accelerate the novel method of the realization of the anatomically correct tissue construct for transplanting.These emerging skills
The set of the art and its synergism action -3D tissues that the nanometer technology of normal physiologic and Pathological Physiology is enabled are imitated by providing
Model-can not only redefine the ability in clinical of regenerative medicine, but also change available for drug discovery and bioscience base
The tool box of plinth research.
Overcome the method for the drawbacks of existing artificial cornea technology undergoes to be to provide a kind of resistance to repel and easily and place
The engineered cornea substitute based on cell of main organizational integration.The present invention relates to such method.
Brief overview
There is provided the cornea substitute that a kind of use 3D biometric prints platform manufactures load cells in the exemplary embodiment
Method and system.Such artificial cornea avoids what is be related in the existing method for the treatment of corneal epithelium disease there is provided a kind of
The new method of many complication.Embodiments in accordance with the present invention, 3D biometric prints machine allows the cell in printing network to encapsulate,
This allows for the biometric print of the institutional framework with micron and nanometer resolution.Carry the cornea substitute of cell
The time that graft is integrated with host tissue can be shortened.Moreover, numeral (that is, customizable) property of 3D printing allows people
The patient-specific tissue model of shape and curvature of the exploitation with design.The cornea tissue of such 3D printing will be in clinic shifting
Plant, mankind's diseases of eye surface models (for example, for scheroma), substitutes or reduce to the early stage drug sieve the need for animal testing
Choose and there is application in time in for the test of the efficacy of drugs of wound healing.
According to exemplary embodiment, by individually cultivating stroma cell living, the endothelial cell (CEC) of work and work
Corneal epithelial cell (CEpC), and the single hypothallus of 3D biometric prints, CEC layers and CEpC layers with by cell encapsulate into individually
Hydrogel nanometer grid in carry out manufacture of intraocular cornea.The first side of hypothallus is attached to by CEC layers and adheres to CEpC layers
To the second side of hypothallus to limit artificial cornea.
In one aspect of the invention, the method for manufacture of intraocular cornea includes culture stroma cell living；3D is biological
Print hypothallus stroma cell living being encapsulated into first hydrogel nanometer grid；Cultivate endothelial cell living
(CEC)；CEC living is encapsulated into the CEC layers in the second hydrogel nanometer grid by 3D biometric prints；Cultivate corneal epithelium living thin
Born of the same parents (CEpC)；CEpC living is encapsulated into the CEpC layers in the 3rd hydrogel nanometer grid by 3D biometric prints；And it is attached by CEC layers
The second side for the first side of hypothallus and being attached to hypothallus by CEpC layers.In certain embodiments, the step of culture
It is parallel progress.Can be with CEC layers and CEpC layers of parallel progress 3D biometric prints the step of.The CEC layers can be by sequentially
Printing hypothallus and CEC layers and be attached to the first side of hypothallus.Alternately, the CEC layers can by between the layers should
Solidify to be attached to the first side of hypothallus with hydrogel thin film and via UV exposures.The CEpC layers can by each layer it
Between exposed using hydrogel thin film and via UV and solidify to be attached to the second side of hypothallus.In a preferred embodiment, exist
Before CEC layers of 3D biometric prints, CEC is mixed with the pre-polymer solution of acryloyl group-polyethylene glycol (PEG)-collagen.Should
Pre-polymer solution may further include the hyaluronic acid (MA-HA) of methacrylated.In another preferred embodiment
In, before CEpC layers of 3D biometric prints, CEpC is mixed with the pre-polymer solution of acryloyl group-PEG- collagens.This is pre-
Copolymer solution may further include MA-HA.In another preferred embodiment, will before 3D biometric print hypothalluses
Stroma cell is encapsulated in the acryloyl group-PEG- collagen hydrogels that may further include MA-HA.These stroma cells can be with
Encapsulated by the cell density that scope is about 5,000,000/ml to 25,000,000/ml stroma cells.
In certain embodiments, cultivated from limbal stem cell (LSC) and break up CEpC living.These LSC can be from certainly
Body tissue is obtained.From the CEC progenitor cells culture from non-human donor and CEC living can be broken up.These CEC progenitor cells can be from
Autologous tissue obtains.
In another aspect of the present invention, artificial cornea includes layer structure, and the layer structure includes 3D biometric prints
Hypothallus (it encapsulates the stroma cell into the work in the first hydrogel nanometer grid), the hypothallus has the first side and the
Two sides；The CEC layers of 3D biometric prints (it encapsulates the CEC into the work in the second hydrogel nanometer grid)；And 3D is biological
The CEpC layers of printing (it encapsulates the CEpC into the work in the 3rd hydrogel nanometer grid)；Wherein the CEC layers is attached to this
First side of hypothallus and the CEpC layers are attached to the second side of the hypothallus.In some embodiments of artificial cornea, CEC
Layer and one or more of the CEpC layers films by application and the hydrogel for exposing and solidifying via UV between the layers are attached
Connect.
Before biometric print hypothallus, preferably stroma cell living is encapsulated into hydrogel.The hydrogel can be with
It is acryloyl group-PEG- collagens, and may further include MA-HA.Before CEC layers of biometric print, also by CEC bags living
Seal into hydrogel.The hydrogel can be acryloyl group-PEG- collagens, and may further include MA-HA.Beaten in biology
Before CEpC layers of print, also CEpC living is encapsulated into hydrogel.The hydrogel can be acryloyl group-PEG- collagens, and
It may further include MA-HA.CEpC living can be obtained from the LSC through cultivating and breaking up.
By the way that emerging technology is incorporated into the multidisciplinary field of nanometer technology, 3D biometric prints and regenerative medicine, we
Artificial cornea has been developed with by eliminating the dependence of current diagonal film donor tissue and being regarded by providing for recovery
The new strategy of power (human patientses otherwise, blinded with severe corneal are by loss of sight) changes clinical scene.Cornea
Primary, multilayer dissection is highly suitable as the original application of our successively nanometer grid conformity 3D printing method.
Brief Description Of Drawings
Fig. 1 is the schematic diagram of the embodiment of 3dLP print systems.
Fig. 2 is the schematic diagram compared using 3D work printing and the embodiment of the artificial cornea of generation with human analogs.
Fig. 3 is the flow chart for manufacturing the illustrative processes of artificial cornea according to embodiments of the present invention.
Fig. 4 A are shown in carries out cell transplantation with the LSC cultivated in the matrix based on gelatin methacrylate (GelMA)
Rabbit corneal afterwards, the rabbit corneal shows that typical corneal epithelial tissue is learned and the smooth and transparent cornea table without Epithelial defects
Face, wherein left small figure shows that H＆E is dyed, and right panel is the white light microphoto of cornea.
Fig. 4 B show the exposed cornea for being just coated with human amniotic membrane, and it shows the histology of epithelial tissue metaplasia and with blood
The opaque cornea of pipe formation.
The rabbit corneal of 3 months after Fig. 4 C display transplanting.
Fig. 5 A-C show the various micro-structurals produced by 3D biometric prints, and wherein Fig. 5 A show having using PEGDA
The multilayer log pile support in 200 μm of apertures；Fig. 5 B show GelMA in 3D printing vascular system sample microstructure (engineer's scale=
30μm)；And Fig. 5 C show the 10T1/2 cells (engineer's scale=1mm) being encapsulated in GelMA supports.
Fig. 6 shows the exemplified synthesis schemes of GelMA hydrogels.
Fig. 7 shows CEC layers of the fusion produced using 3dLP systems.
Fig. 8 A-C show the assessment of the optical property of the aquagel membrane with different component.
Fig. 9 A-9C be respectively displayed on the definition of the 5th day, the 10th day and the 15th day corneal transplant and function after transplanting by
Gradually recover.
Figure 10 is the stream for designing, manufacturing and transplanting the illustrative processes of artificial cornea according to an embodiment of the invention
Cheng Tu.
Detailed description of the invention
By the way that emerging technology is incorporated into the multidisciplinary field of nanometer technology, 3D biometric prints and regenerative medicine, we
Artificial cornea has been developed with by eliminating the dependence of current diagonal film donor tissue and being regarded by providing for recovery
The new strategy of power (human patientses otherwise, blinded with severe corneal are by loss of sight) changes clinical scene.The present invention
Method utilize the 3D printing based on nanometer be used for cornea regeneration.The primary of cornea, multilayer dissection are highly suitable as ours
The successively original application of nanometer grid conformity 3D printing method.
3D work printing (" 3dLP ") technologies are projected and automated platform by digital micro-mirror device (DMD) to utilize series of layers
Continuous 3D printing.Previously disclosed similar 3D printing system for different applications.(see, for example, International Publication
Number WO 2014/197622 and international publication number WO 2012/071477, is incorporated herein by reference).
Printing is projected using digital mask (that is, " maskless ") using 3D hydrogel matrix manufacture of intraocular cornea, wherein
The digital micro-mirror device (DMD) that is found in conventional computer projecting apparatus using ultraviolet (UV) or be suitable for selected polymer its
His light source polymerize and solidified sensitive liquid prepolymer.Fig. 1 shows to be referred to as " dynamic projection photocureable rapid shaping " (DPsL)
The maskless of platform projects the example implementations of print system 2.Should " maskless " or digital mask method allow using can
Control and interchangeable reflected light pattern, rather than static, more expensive physical mask as used in conventional lithographic.
System 2 includes UV light sources 6, the computer control 10 that the sheet image stream for instructing pattern to produce is generated, by about 100
Dmd chip 12 (it is embedded in projecting apparatus as dynamic mask) that ten thousand micro mirrors are constituted, projection optical device 14, for sample bit
Put the translation stage 16 of control and the source of photocurable prepolymer material 13.The dmd chip 12 is served as installed in small hinge
On the array for covering aluminium reflective micro-mirrors, the hinge enables these micro mirrors towards light source or away from light source incline, in projection table
Light ("ON") or dark ("Off") pixel are produced on face, so as to allow its light for redirecting two states [0,1], with two biass
Electrode incline is to form relative to+12 ° of surface or -12 ° of angles.By this way, DMD systems can reflect up to 1,024 kind
The pixel of gray scale with generate height in detail gray level image.
Computer control 10 can be shown to the image (as shown in the figure) of the hope structure 8 of given layer and/or can show
The hope parameter of matrix.Quartz window or other light transmissive materials 15, sept 18 and substrate 19 are supported on translation stage 16, it
Define the print volume comprising pre-polymer solution 13 or " father-in-law (vat) ".Syringe pump (not shown) can be used as needed
Other solution 13 is introduced into print volume.Based on the order generated by controller 10, the system uses dmd chip 12
(1920 × 1080 resolution ratio) carries out spatial modulation to parallel UV light, and customized optical design is projected into photocurable
Pre-polymer solution 13 on.
In order to produce 3D structures, projection photocurable rapid shaping platform (for example, DPsL) is using successively fabrication schedule.Showing
In example property method, 3D computers perspective view (being made of CAD software or CT scan) is deconstructed into a series of evenly spaced planes
Or layer.For purposes of illustration, the latticed knot represented desired by one layer is illustrated on the display 8 of computer control 10
The simple honeycomb pattern of structure.Every layer of pattern is input into dmd chip 12, so that it is (pre- that UV light is exposed into photocurable
Polymers) on material 13 to produce polymer architecture 17.Formed to one layer after pattern, the reduction automation of computer control 10
Platform 16, and show next pattern to build the height of polymer architecture 17.By being programmed to computer control 10,
User can be with the height of control platform speed, luminous intensity and structure 17, so as to allow to manufacture various labyrinths 20.It should note
Meaning, although show single honeycomb, but any combinations of pattern can be used to construct different pattern superposed on one another
Sandwich construction.
As an alternative scheme of dmd chip, galvanometric optical scanner or polygon scanning mirrors can be used.This two
Kind of technology is commercially available, and their applications in quick scanning confocal microscope are known.Selection is a kind of appropriate
Scan mechanism to be used in combination within the level of those skilled in the art with the system and method for the present invention.
According to exemplary embodiment, 3 step strategies are followed for manufacturing the method for the artificial cornea based on cell.With reference to Fig. 3,
In the step 32, we set up and optimize be embedded with the basilar memebrane of nanometer grid grow CEpC (corneal epithelial cell) and
CEC (endothelial cell) condition of culture.It is determined that after optimal culture condition, according to the successively side in our 3dLP systems
Case, we are lived using 3D prints three vitreous layers of assembling.In step 34, by stroma cell using scope as about 5,000,000/ml extremely
(7.5wt% adds 25wt% to the Ac-Col hydrogels that the cell density of 25000000/ml stroma cells is encapsulated in similar to primary cornea
PEGDA) in (acryloyl group-PEG- collagens).Projection time for printing this layer can be between 1 second to 5 seconds.In step 36
In, by via in the nanometer grid of 3D nanometers of printing manufactures simultaneously embedded hypothallus.Use the optimal conditions from step 32, warp
CEC layers and CEpC layers are assembled with matrix by two parallel schemes：It is in step 38 and 40, CEC and Ac-Col prepolymers is molten
Liquid (5wt%) is mixed and printed to via photopolymerization together with nanometer grid on hypothallus within 30 seconds., can be with step 42 and 44
The CEpC layers on the opposite side of matrix are printed upon using similar method.CEC layers and CEpC layers need not simultaneously or sequentially beat
It is imprinted on the opposite side of hypothallus.Alternately, the CEC layers developed in advance are with CEpC layers (in its respective incorporation nanometer grid
Basilar memebrane on have fusion cellular layer) can by between the layers application Ac-Col films and via UV exposure and
Solidification comes " gluing " to (step 46) in matrix.The construct finally printed is fully rinsed any to eliminate with brine buffer solution
The unpolymerized solution (step is not shown) of residual, and further keep in the medium until transplanting.Finally, these 3D printings
Cornea be ready for transplanting and functional assessment.
Following example provide the details for the step of using in an embodiment of the present invention：
Example 1：CEpC, CEC and stroma cell are grown on basilar memebrane
Corneal epithelial cell (CEC) is undergone from the cyclic regeneration of limbal stem cell or progenitor cells (LSC) and in LSC
Or the defect in corneal epithelium, cornea is become opaque keratinized skin sample epithelium by it, and causing causes the cornea table of blindness
Face disease.How LSC maintains and is divided into corneal epithelium in healthy individuals, and which molecular events is defective in patients
It is largely unknown.
Traditionally, LSC growths and amplification procedure need mouse 3T3 feeder cells, and it carries the pollution from animal product
Risk, therefore make it unsuitable for producing the cornea of clinically feasible 3D biometric prints.In order to overcome these obstacles, develop
It is a kind of it is external without feeder cells, chemically defined cell culture systems to grow the LSC from rabbit and non-human donor, to cause
LSC homogeneous population can be produced and expanded, and be subsequently differentiate into corneal epithelial cell (CEpC).The culture systems be based on
It is lower to determine：Transcription factor p63 (oncoprotein 63) and PAX6 (paired box protein PAX6) are acted as be used to specify LSC together, and
WNT7A controls cornea epithelial differentiation by PAX6.In limbal stem cell, WNT7A acts on PAX6 upstream, and via
Frizzled homologue 5 (FZD5) (acceptor of WNT protein) stimulates it to express.WNT7A be secretion morphogen, its participate in developmental character and
Pathogenic WNT signal transductions.PAX6 is the transcription factor for the destiny and differentiation for controlling various ocular tissues.The WNT7A of RNAi mediations
Or PAX6 knockout induces human limbal stem cell to be transformed into skin epidermis form, a kind of and common human corneal disease from cornea
The critical defect that disease is closely related.WNT7A and PAX6, which knocks out cell, also has the cornea angle lower than wild type corneal limbal cells
(the KRT3 of albumen 3；CK3) and KRT12 expression and bigger skin epidermis KRT1 and KRT10 expression.
It is interesting to note that transductions of the PAX6 in skin epidermis stem cell is enough to convert them into LSC like cells, and
When on the eyes being transplanted in rabbit corneal damage model, these cells reprogramed can supplement CEC and repair damage
Anterior corneal surface.In the bulletin being described in further detail in disclosed in documents below of this method：《It is natural》(Nature), " WNT7A
Corneal epithelium homeostasis and pathogenesis (WNT7A and PAX6define Corneral epithelium are limited with PAX6
Homeostatis and pathogenesis) ",《It is natural》(2014)doi:10.1038/nature13465, in 2014 7
The moon 2 is online open, is incorporated herein by reference.Proliferative LSC is characterised by P63 and K19 expression, high percentage
Dyeing is positive to mitotic marker Ki67.We establish 3D LSC Differentiation Systems, wherein the CEpC layers being layered are similar
Grown in the basilar memebrane of bag Man (Bowman ' s) film.Small molecule-ROCK inhibitor Y27632 is used to instruct LSC to be divided into
CEpC, being proved such as the strongly expressed by CEpC specific markers K3/K12.
Abreast, we have developed without feeder cells, chemically defined cell culture systems, the cell culture system contains
There is FGF2 (FGF2) to grow the CEC progenitor cells from non-human donor.Then by these CEC progenitor cells
The homogeneous population of CEC progenitor cells is expanded into, the homogeneous population is subsequently differentiate into CEC.It is observed that being present in primary dissection
Hexagon form, with typical CEC mark ZO-1 strongly expressed.
It is possibly used for moreover, we test the LSC cultivated in the matrix based on gelatin methacrylate (GelMA)
Treatment and the potential for repairing the corneal epithelial defects in the rabbit LSC defect models of the common disease of cornea situation of the simulation mankind.
In this test, the epithelium that the LSC grafts that rabbit GFP is marked form the positive staining with cornea specificity K3/12 is thin
The continuous slice of born of the same parents, and the Epithelial defects of whole anterior corneal surface have successfully been repaired, and recover and maintain cornea definition
With transparency more than 5 months.
Fig. 4 A-4C show the result of these tests：Fig. 4 A are shown with the GFP marks cultivated in the matrix based on GelMA
The LSC of note carries out the rabbit corneal after cell transplantation, and the rabbit corneal shows that (Zuo little Tu learns in typical corneal epithelial tissue：H＆E contaminates
Color) and smooth and transparent anterior corneal surface (right panel without Epithelial defects：White light microphoto).Fig. 4 B, which are shown, to be just coated with
The exposed cornea of human amniotic membrane.Zuo little Tu shows the histology of epithelial tissue metaplasia, and right panel is shown with vascularization not
Transparency cornea.Trimestral smooth, transparent rabbit corneal after Fig. 4 C display transplanting.By in the grown on matrix based on GelMA
Culture GFP+LSC be used for transplantation experiments in, wherein they are dyed to show that it is whole with recipient cornea epithelium altogether with K3/12
Close.
Also cultivate and expand in vitro keratocyte.These stroma cells share fibroblastic similar mark,
Such as fibronectin, FSP1 and vimentin.
Example 2：3D biometric prints
3D biometric prints platform provides the rapid biologic manufacture method for the hydrogel scaffold for being used to build carrying cell, should
Support 1) there is the complicated user-defined 3D geometries being made up of the biomaterial of natural origin；2) allow to be encapsulated in water
The consistent 3D distributions of cell in gel；3) sertoli cell vigor and propagation；And 4) it is characterized as dynamic, multiple dimensioned mechanics
Cell-scaffold interaction.Importantly, these constructs enable control over and integrated the 3D geometries of complexity, together
When the related inside 3D distributions of physiology of encapsulation cell are provided.It is distributed by the room and time to biotic factor in 3D supports
This accurate control, we can assess phase interaction of the cell with extracellular matrix (ECM) albumen on nanometer length scale
It is to create advanced, clinically transferable biomimetic scaffolds with, its final goal.
Using 3D biometric prints, using manufacture of intraocular cornea is carried out with primary cornea identical size and curvature to replicate patient
Cornea.The material of natural origin can support the cell growth in construct and restore host cell with preferably construction and integration
Body.Due to the high efficiency of 3D printing technique, just it is enough within several seconds to complete one layer.Therefore, the thin of height homogeneous is kept in every layer
Born of the same parents' distribution is possible.Furthermore, it is possible to accurately control the space orientation of different cell types, this is the key of cornea function.Example
Such as, we can manufacture the small feature of about 5 microns (that is, less than cell).Using this resolution ratio, we can control very small
Cell colony, or even individual cells space orientation.By using the material of different degraded overviews, we can instruct cell
Migration, so as to control its Annual distribution.By the patterned growth factor in construct, we can also adjust cell propagation/
Differentiation, and manage cell distribution.
Fig. 5 A-C show the exemplary microstructure produced by 3D biometric prints：Fig. 5 A, have 200 μ using PEGDA
The multilayer round log shape support in m apertures；The vascular system sample microstructure (engineer's scale=30 μm) of 3D printing in Fig. 5 B, GelMA；
Fig. 5 C, the 10T1/2 cells being encapsulated in GelMA supports keep vibrant and breed in 8 hours after encapsulating, via calcein-
The survival of AM/ second ingot homodimers/death is determined and is estimated (engineer's scale=1mm).
Example 3：Biomaterial for cornea tissue
Collagen has been widely used as the biomaterial of cornea histoengineering, because it includes corneal extracellular matrix
(ECM) key component.Have been demonstrated to support epithelial cell formation protective layer as the collagen of matrix components and promote to pass through
The laryngeal muscles of neuron.The biosynthesis collagen-based matter of chemical crosslinking is shown significantly in Phase I clinical trial
Prospect.In order to adjust degraded and the mechanical performance of collagen stroma, most of researchs use Chemical Crosslinking Methods, these chemistry
Cross-linking method is largely encapsulated incompatible with cell.Acryloyl group-PEG- collagens (Ac-Col) are due to its bio-compatible
Property, optical property and photopolymerization ability and fabulous substitute is provided for cornea histoengineering.Make a preliminary test
Optical property to assess the film by GelMA (it is Ac-Col analog) the texture cells being made.Fig. 6 shows GelMA
The exemplified synthesis schemes of hydrogel.CEC is inoculated with and cultivated in the optical clear cornea manufactured with GelMA using 3dLP systems
In matrix.Even after the CEC cell sheets (being shown in Fig. 7) of fusion are formed, the transparency of construct is maintained.
Influence to different hybridization hydrogel combination and open-assembly time to optical clarity is evaluated.Fig. 8 A-8C
Show the result for the optical clarity for comparing the UCSD marks by manufactured structure observation for every kind of combination.Fig. 8 A exhibitions
7.5wt%GelMA (the gelatin methyl with 1wt%MA-HA (methacrylate-hyaluronic acid) (MW=200KDa) is shown
Acrylate) reduction transparency, UV expose=1 minute.As shown in Figure 8 B, with 7.5wt%GelMA, 1wt%MA-HA (MW
=200KDa) and 2.5%PEGDA (PEG diacrylate) (MW=700KDa) realize the improvement of transparency, UV
Exposure=30 seconds.Use 7.5wt%GelMA, 2.5wt%MA-HA (MW=200KDa) and 2.5%PEGDA (MW=700KDa)
More preferable transparency is still obtained with UV exposures within=30 seconds.These results indicate that as MA-HA concentration increases to from 1wt%
2.5wt%, definition increase.
Different materials composition has been tested, and the optical property of most of materials selection is very good.In a reality
In example, add 0.075wt%LAP (phenyl -2,3,6- trimethylbenzene first with 7.5wt%GelMA or Ac-Col and 25wt%PEGDA
Acyl group hypophosphorous acid lithium) as light trigger, generate suitable with PBS solution displaying suction in the range of 280nm to 1000nm
The hyaline membrane of luminosity.UV open-assembly times seem not influence the transparency of the film.For MA-HA, with 2.5wt%MA-HA and
2.5%PEGDA 7.5wt%GelMA also provides fabulous optical property after the UV exposures of 30 seconds.
As known in the art, because most of light triggers are cytotoxicities.Select the type of light trigger and dense
Spend to obtain desired film character, while keeping cell viability will be in art technology level.
Example 4：The transplanting of the cornea of 3D printing
Printing is lived using 3D as described above in manufacturing three vitreous layers.Specifically, PEGDA nanometers of grids are embedded in propylene
To support corneal stroma in acyl group-PEG- collagens.CEpC layers and CEC layers are set up in every side of hypothallus.By the life of gained
The corneal transplantation of thing printing is on rabbit recipient eye.
It is by intramuscular injection xylazine hydrochloride (2.5mg/ml) and ketalar (37.5mg/ml) that NZw is numb
It is liquor-saturated.The cornea receiver substrate bed with reverse button spline structure is produced in acceptor eye using femtosecond laser machine (Zeiss).Will be raw
The corneal donor tissue of thing printing cuts into button-type structure to be fitted on the receiver substrate bed of preparation.Then mankind sheep is passed through
Film (company of biological tissue (Bio-tissue)) covers surface, and with 10.0VICRYL sutures (surface is treasured into Kanggong department
(Ethicon)) it is fixed on recipient conjunctival.Fig. 9 A and 9B are respectively displayed on the 5th day and the 10th day definition and function after transplanting
Gradually recovery.Observe within the 15th day the gradually increase gradually decreased with cornea definition of corneal edema (as schemed after the transfer
Shown in 9C), this shows the functional rehabilitation of corneal endothelium.It was observed that anterior corneal surface epithelium is smooth and complete, this shows work(
The CEpC of energy property transplanting.
According to embodiment as described herein, the use of 3D biometric print technologies allows cell to encapsulate, enabling printing living
Institutional framework with micron and nanometer resolution.It is whole with host tissue that the cornea substitute of carrying cell can reduce graft
Time quantum needed for closing.In addition, numeral (that is, customizable) property of 3D printing allows shape and curvature of the exploitation with design
Patient-specific tissue model.Can be according to the shape and curvature of the primary cornea design customization of patient.
Using program known in the art, corneal topography can be obtained for patient before transplanting program and measured.For example,
The instrument used in clinical practice is most often based on placido (Placido) Reflected Image Analyzer, and it is using to being incident upon cornea
On the analyses of reflected image of multiple concentric rings obtain the dioptric scope and surface curvature of measurement of corneal astigmatism.Using passing through
The clinical data that this test is produced, computer software can be used for producing patient-specific cornea design, then will use 3D
Print platform designs to manufacture the cornea.Successively Method of printing can be used.In some cases, it is highly complex in order to produce
Cornea geometry, can be suitably with non-linear 3D printing scheme, the PCT application for example submitted for 16th in September in 2015
The scheme disclosed in number PCT/US 2015/050522, this application is incorporated herein by reference.
Figure 10 summarizes design, manufacture and the exemplary journey of transplanting for artificial cornea according to an embodiment of the invention
Sequence.Replacement to determine cornea is medically required beginning, in step 50, uses the clinical instrument for measuring patient's cornea
Device produces data.Using computer aided design software, in step 52, develop a series of printing steps to control 3dLP to beat
Artificial cornea is fabricated to the just size and desired feature of patient's eye by print machine.It is used to print patient-specific with creating
Stroma cell and LSC concurrently, are cultivated and to be mixed into prepolymer molten by the computer-controlled program of cornea in step 60 to 67
In liquid.While not limited to using patient itself cell, but use autologous tissue as stroma cell, progenitor cells CEC, and/or
LSC source can provide additional advantage, that is, reduce or eliminate to immunosuppressive possible demand.In step 63 and 66,
LSC is divided into CEpC respectively, and the CEC progenitor cells from non-human donor is divided into CEC.In step 61,64 and 67,
The cell of culture is each mixed into pre-polymer solution.(it should be noted that, although flow chart is shown in form CEC layers and CEpC
Hypothallus is prepared before layer, but this one or more of three layers can print in different time, for example, print in advance, or
They can be by parallel printing, i.e. is not to print in a particular order, and assembles as described above.) in step 54, will
Stroma cell, CEC and the CEpC of culture are mixed in its respective layer as described above.They can sequentially be printed or individually
Ground is printed, and assembles to limit the CEC- matrix-CEpC hierarchies of cornea from the layer individually printed.Use at step 56
Program known in the art removes defective cornea, and prepares matrix bed to receive graft, then moves in step 58
Plant artificial cornea.
The cornea tissue of the 3D printing manufactured according to program as described herein will be modeled in clinical transplantation, human eye surface's disease
(for example, for scheroma), substitute or reduce to being cured in the early stage drug screening the need for animal testing and for wound
There is application in time in the efficacy of drugs test of conjunction.This technology provides solid for the exploitation of temporarily or permanently cornea substitute
Basis.Embodiment as described herein can cause the complicated engineering tissue being readily available, and it is natural that these tissues reappear its
The function of mankind's homologue and it is suitable for clinical practice and emerging biomedical research.
Bibliography (is incorporated herein by reference)
1. take Ge Huoermu (Fagerholm) P, La Jiali (Lagali) NS, Mei Leite (Merrett) K, Jackson
(Jackson) WB, illiteracy gill (Munger) R, Liu (Liu) Y, ripple thunder rake (Polarek) JW, Suo De Qwests
(Soderqvist) M and Griffith (Griffith) M, the biological conjunction that the non-human donor for inducing cornea regeneration organizes
Into substitute：(the A biosynthetic alternative to human donor of follow-ups in 24 months of 1 phase clinical research
tissue for inducing corneal regeneration:24-month follow-up of a phase 1
Clinical study), science translational medicine (Sci Transl Med.) 2010；2(46):46ra61
Mu Yang 2. (Myung) D, Du breathe out Mel (Duhamel) PE, Cochran (Cochran) JR, Nolan's enlightening (Noolandi)
J, tower (Ta) CN and Frank (Frank) CW, the exploitation of the corneal prostheses based on hydrogel：Material visual angle
(Development of hydrogel-based keratoprostheses:A materials perspective), it is biological
Technical progress (Biotechnol Prog) 2008；24(3):735-41
3. carat primary (Crabb) RA, week (Chau) EP, Evans (Evans) MC, this (Barocas) VH of Barrow card and
Xiu Boer (Hubel) A, the biomethanics and micro-structural feature of the corneal stroma equivalent based on collagem membrane
(Biomechanical and microstructural characteristics of a collagen film-based
Corneal stroma equivalent), organizational engineering (Tissue Eng.) 2006；12(6):1565-75.
Claims (30)
1. a kind of method for manufacture of intraocular cornea, this method includes：
Cultivate stroma cell living；
3D biometric print hypothalluses, the hypothallus encapsulates stroma cell living into the first hydrogel nanometer grid；
Cultivate endothelial cell (CEC) living；
CEC layers of 3D biometric prints, the CEC layers is encapsulated CEC living into the second hydrogel nanometer grid；
Cultivate corneal epithelial cell (CEpC) living；
CEpC layers of 3D biometric prints, the CEpC layers is encapsulated CEpC living into the 3rd hydrogel nanometer grid；And
The first side for being attached to the hypothallus by the CEC layers, and the second side for being attached to the hypothallus by the CEpC layers.
2. the method as described in claim 1, wherein the step of abreast being cultivated.
3. the method as described in claim 1, wherein abreast carrying out the step of CEC layers of 3D biometric prints are with the CEpC layers.
4. the method as described in claim 1, wherein the step of being attached to the first side of the hypothallus by the CEC layers includes order
Ground prints the hypothallus and the CEC layers.
5. the method as described in claim 1, wherein the step of being attached to the first side of the hypothallus by the CEC layers is included each
Solidified between layer using hydrogel thin film and via UV exposures.
6. the method as described in claim 1, wherein the step of being attached to the second side of the hypothallus by the CEpC layers includes
Solidified between each layer using hydrogel thin film and via UV exposures.
7. the method as described in claim 1, this method further comprises before the CEC layers of 3D biometric prints, by these CEC
Mixed with the pre-polymer solution of acryloyl group-PEG- collagens.
8. method as claimed in claim 7, the wherein pre-polymer solution further comprise MA-HA.
9. the method as described in claim 1, this method further comprises before the CEpC layers of 3D biometric prints, by these
CEpC is mixed with the pre-polymer solution of acryloyl group-PEG- collagens.
10. method as claimed in claim 9, the wherein pre-polymer solution further comprise MA-HA.
11. the method as described in claim 1, this method further comprises before the 3D biometric prints hypothalluses, by these
Stroma cell is encapsulated in acryloyl group-PEG- collagen hydrogels.
12. method as claimed in claim 11, the wherein pre-polymer solution further comprise MA-HA.
13. method as claimed in claim 11, wherein using the cell of scope stroma cell as about 5,000,000/ml to 25,000,000/ml
Density encapsulates these stroma cells.
14. the method as described in claim 1, wherein the step of cultivating CEpC living includes culture LSC, and breaks up LSC
Into CEpC.
15. method as claimed in claim 14, wherein these LSC are obtained from autologous tissue.
16. the method as described in claim 1, wherein the step of cultivating CEC living is including cultivating the CEC ancestrals from non-human donor
Cell, and CEC progenitor cells is divided into CEC.
17. method as claimed in claim 14, wherein these CEC progenitor cells are obtained from autologous tissue.
18. the method as described in claim 1, wherein first, second, and third hydrogel nanometer grid include PEGDA.
19. a kind of artificial cornea, the artificial cornea includes：
A kind of layer structure, the layer structure includes：
The hypothallus of 3D biometric prints, the hypothallus is encapsulated in the stroma cell of the work in the first hydrogel nanometer grid,
The hypothallus has the first side and the second side；
The CEC layers of 3D biometric prints, the CEC layers be encapsulated in the second hydrogel nanometer grid in work endothelial cell
(CEC)；
The CEpC layers of 3D biometric prints, the CEpC layers be encapsulated in the 3rd hydrogel nanometer grid in work corneal epithelium it is thin
Born of the same parents (CEpC)；And
The first side for being wherein attached to the hypothallus by the CEC layers, and the second side for being attached to the hypothallus by the CEpC layers.
20. artificial cornea as claimed in claim 19, wherein the CEC layers and one or more of the CEpC layers passes through application
It is attached between the layers and via the UV hydrogel thin films for exposing and solidifying.
21. artificial cornea as claimed in claim 19, wherein before the biometric print hypothallus, the matrix of these work is thin
Born of the same parents are encapsulated in hydrogel.
22. artificial cornea as claimed in claim 21, the wherein hydrogel include acryloyl group-PEG- collagens.
23. artificial cornea as claimed in claim 20, the wherein hydrogel further include MA-HA.
24. artificial cornea as claimed in claim 19, wherein before the CEC layers of biometric print, the CEC of these work is encapsulated
In hydrogel.
25. artificial cornea as claimed in claim 24, the wherein hydrogel include acryloyl group-PEG- collagens.
26. artificial cornea as claimed in claim 25, the wherein hydrogel further include MA-HA.
27. artificial cornea as claimed in claim 19, wherein these CEpC lived include the LSC through cultivating and breaking up.
28. artificial cornea as claimed in claim 19, wherein before the CEpC layers of biometric print, by the CEpC bags of these work
It is enclosed in hydrogel.
29. artificial cornea as claimed in claim 27, the wherein hydrogel include acryloyl group-PEG- collagens.
30. artificial cornea as claimed in claim 28, the wherein hydrogel further include MA-HA.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US201462054924P | 2014-09-24 | 2014-09-24 | |
| US62/054,924 | 2014-09-24 | ||
| PCT/US2015/051999 WO2016049345A1 (en) | 2014-09-24 | 2015-09-24 | Three-dimensional bioprinted artificial cornea |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107106734A true CN107106734A (en) | 2017-08-29 |
Family
ID=55582016
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201580062303.9A Pending CN107106734A (en) | 2014-09-24 | 2015-09-24 | The artificial cornea of three dimensional biological printing |
Country Status (5)
| Country | Link |
|---|---|
| US (1) | US20170281828A1 (en) |
| JP (1) | JP2017529842A (en) |
| CN (1) | CN107106734A (en) |
| RU (1) | RU2017111686A (en) |
| WO (1) | WO2016049345A1 (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109701083A (en) * | 2019-01-25 | 2019-05-03 | 上海交通大学医学院附属第九人民医院 | It is a kind of to prepare artificial tendon method using biological 3 D-printing and electrostatic spinning technique |
| CN109718397A (en) * | 2017-10-31 | 2019-05-07 | 湖南大学 | A kind of biological micro-assembly robot of assembling |
| CN110101916A (en) * | 2019-05-17 | 2019-08-09 | 张勇 | Synthesize cornea and its preparation method and application |
| WO2021012534A1 (en) * | 2019-07-23 | 2021-01-28 | 清华大学深圳国际研究生院 | Micro-fluidic chip printing nozzle and biological 3d printing system |
| CN114763399A (en) * | 2021-02-05 | 2022-07-19 | 天津大学 | Digital light processing 3D printing artificial cornea, printing method and application thereof |
| CN115671398A (en) * | 2022-11-22 | 2023-02-03 | 首都医科大学附属北京同仁医院 | 3D printing bionic corneal limbus graft and preparation method and application thereof |
| WO2023123307A1 (en) * | 2021-12-27 | 2023-07-06 | 暨南大学 | Living cell bionic corneal anterior lamellar and preparation method therefor |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20210187161A1 (en) * | 2016-04-14 | 2021-06-24 | Massachusetts Eye And Ear Infirmary | Medical device comprising graphene coating |
| US10738297B2 (en) | 2016-05-13 | 2020-08-11 | University Of Washington | 3D printable hydrogel materials |
| US10073346B2 (en) | 2016-07-08 | 2018-09-11 | Cypre, Inc. | Apparatus for patterning hydrogels into multi-well plates |
| CA3043194A1 (en) | 2016-11-09 | 2018-05-17 | The United States Of America, As Represented By The Secretary, Department Of Health & Human Services | 3d vascularized human ocular tissue for cell therapy and drug discovery |
| WO2018089521A1 (en) | 2016-11-09 | 2018-05-17 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Tissue clamp and implantation method |
| US11085018B2 (en) | 2017-03-10 | 2021-08-10 | Prellis Biologics, Inc. | Three-dimensional printed organs, devices, and matrices |
| US10933579B2 (en) | 2017-03-10 | 2021-03-02 | Prellis Biologics, Inc. | Methods and systems for printing biological material |
| EP3635105A4 (en) | 2017-05-25 | 2021-03-31 | Prellis Biologics, Inc. | Three-dimensional printed organs, devices, and matrices |
| KR102114787B1 (en) * | 2018-04-02 | 2020-05-26 | 중앙대학교 산학협력단 | Preparing method for 3 dimension corneal endothelial graft comprising ribonuclease 5 overexepressing corneal endothelial cells |
| EP3829850A1 (en) | 2018-07-28 | 2021-06-09 | Calt Dynamics Ltd | Methods, systems, and devices for three-dimensional object generation and physical mask curing |
| US11235515B2 (en) * | 2018-07-28 | 2022-02-01 | CALT Dynamics Limited | Methods, systems, and devices for three-dimensional object generation and physical mask curing |
| WO2021133457A2 (en) * | 2019-10-08 | 2021-07-01 | Massachusetts Eye And Ear Infirmary | Ocular sealants and methods of using the same |
| GB202001285D0 (en) * | 2020-01-30 | 2020-03-18 | Univ Newcastle | Novel tissues and methods of making the same |
| TR202020549A2 (en) * | 2020-12-15 | 2022-06-21 | Istanbul Medipol Ueniversitesi | A LIFE CELL-LOADED POLYMER/ENzyme HYDROJEL METHOD AND AN ARTIFICIAL CORNEA LAYER MANUFACTURED BY THE SUCH METHOD |
| CN113456895B (en) * | 2021-07-20 | 2022-05-06 | 广州市朴道联信生物科技有限公司 | GelMA-collagen double-network antibacterial cornea repair material and preparation method and application thereof |
| EP4227399A1 (en) | 2022-02-14 | 2023-08-16 | Fundación para la Investigación Biomédica del Hospital Universitario de la Paz | Artificial constructs for use in ophthalmology, methods of obtaining the same, and their use |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1473551A (en) * | 2003-08-07 | 2004-02-11 | 中山大学中山眼科中心 | Artificial tissue engineeing biological cornea |
| US20060083773A1 (en) * | 2004-10-05 | 2006-04-20 | David Myung | Artificial corneal implant |
| US20110212501A1 (en) * | 2008-09-12 | 2011-09-01 | The Brigham And Women's Hospital, Inc. | 3-dimensional multi-layered hydrogels and methods of making the same |
| WO2011154687A1 (en) * | 2010-06-11 | 2011-12-15 | Ucl Business Plc | Biomimetic corneal tissue |
| US20130190210A1 (en) * | 2011-09-12 | 2013-07-25 | Organovo, Inc. | Engineered tissues for in vitro research uses, arrays thereof, and methods of making the same |
| CN104768586A (en) * | 2012-09-04 | 2015-07-08 | 人类起源公司 | Methods of tissue generation |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008066965A2 (en) * | 2006-06-23 | 2008-06-05 | The Regents Of The University Of California | Articles comprising large-surface-area bio-compatible materials and methods for making and using them |
| WO2013184843A1 (en) * | 2012-06-05 | 2013-12-12 | The Regents Of The University Of California | Novel methods to regenerate human limbal stem cells |
-
2015
- 2015-09-24 CN CN201580062303.9A patent/CN107106734A/en active Pending
- 2015-09-24 RU RU2017111686A patent/RU2017111686A/en not_active Application Discontinuation
- 2015-09-24 JP JP2017515772A patent/JP2017529842A/en active Pending
- 2015-09-24 WO PCT/US2015/051999 patent/WO2016049345A1/en active Application Filing
- 2015-09-24 US US15/513,938 patent/US20170281828A1/en not_active Abandoned
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1473551A (en) * | 2003-08-07 | 2004-02-11 | 中山大学中山眼科中心 | Artificial tissue engineeing biological cornea |
| US20060083773A1 (en) * | 2004-10-05 | 2006-04-20 | David Myung | Artificial corneal implant |
| US20110212501A1 (en) * | 2008-09-12 | 2011-09-01 | The Brigham And Women's Hospital, Inc. | 3-dimensional multi-layered hydrogels and methods of making the same |
| WO2011154687A1 (en) * | 2010-06-11 | 2011-12-15 | Ucl Business Plc | Biomimetic corneal tissue |
| US20130190210A1 (en) * | 2011-09-12 | 2013-07-25 | Organovo, Inc. | Engineered tissues for in vitro research uses, arrays thereof, and methods of making the same |
| CN104768586A (en) * | 2012-09-04 | 2015-07-08 | 人类起源公司 | Methods of tissue generation |
Non-Patent Citations (2)
| Title |
|---|
| XU FENGLAN ET AL: "《Preparation and in vivo investigation of artificial cornea made of nano-hydroxyapatite/poly (vinyl alcohol) hydrogel composite》", 《J MATER SCI:MATER MED》 * |
| YI LU ET AL: "《A digital micro-mirror device-based system for the microfabrication of complex, spatially patterned tissue engineering scaffolds》", 《JOURNAL OF BIOMEDICAL RESEARCH MATERIALS》 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109718397A (en) * | 2017-10-31 | 2019-05-07 | 湖南大学 | A kind of biological micro-assembly robot of assembling |
| CN109718397B (en) * | 2017-10-31 | 2022-03-15 | 湖南大学 | Assembled biological micro-tissue |
| CN109701083A (en) * | 2019-01-25 | 2019-05-03 | 上海交通大学医学院附属第九人民医院 | It is a kind of to prepare artificial tendon method using biological 3 D-printing and electrostatic spinning technique |
| CN110101916A (en) * | 2019-05-17 | 2019-08-09 | 张勇 | Synthesize cornea and its preparation method and application |
| CN110101916B (en) * | 2019-05-17 | 2019-12-31 | 张勇 | Synthetic cornea and preparation method and application thereof |
| WO2021012534A1 (en) * | 2019-07-23 | 2021-01-28 | 清华大学深圳国际研究生院 | Micro-fluidic chip printing nozzle and biological 3d printing system |
| CN114763399A (en) * | 2021-02-05 | 2022-07-19 | 天津大学 | Digital light processing 3D printing artificial cornea, printing method and application thereof |
| CN114763399B (en) * | 2021-02-05 | 2023-07-07 | 天津大学 | Digital light processing 3D printing artificial cornea, printing method and application thereof |
| WO2023123307A1 (en) * | 2021-12-27 | 2023-07-06 | 暨南大学 | Living cell bionic corneal anterior lamellar and preparation method therefor |
| CN115671398A (en) * | 2022-11-22 | 2023-02-03 | 首都医科大学附属北京同仁医院 | 3D printing bionic corneal limbus graft and preparation method and application thereof |
| CN115671398B (en) * | 2022-11-22 | 2024-03-01 | 首都医科大学附属北京同仁医院 | 3D printing bionic limbal implant and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2016049345A1 (en) | 2016-03-31 |
| US20170281828A1 (en) | 2017-10-05 |
| JP2017529842A (en) | 2017-10-12 |
| RU2017111686A (en) | 2018-10-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107106734A (en) | The artificial cornea of three dimensional biological printing | |
| Yu et al. | Photopolymerizable biomaterials and light-based 3D printing strategies for biomedical applications | |
| Zhu et al. | Rapid continuous 3D printing of customizable peripheral nerve guidance conduits | |
| Ng et al. | Vat polymerization-based bioprinting—process, materials, applications and regulatory challenges | |
| Warner et al. | Design and 3D printing of hydrogel scaffolds with fractal geometries | |
| Grogan et al. | Digital micromirror device projection printing system for meniscus tissue engineering | |
| Wilson et al. | Control of scar tissue formation in the cornea: strategies in clinical and corneal tissue engineering | |
| CN105209605B (en) | It is engineered hepatic tissue, its array and preparation method thereof | |
| US20220025322A1 (en) | Compositions and methods for printing three-dimensional structures corresponding to biological material | |
| US20060287721A1 (en) | Artificial cornea | |
| CN109157305B (en) | Composite artificial cornea and preparation method thereof | |
| WO2021146466A1 (en) | Methods and systems for model generation | |
| Hockaday et al. | 3D-printed hydrogel technologies for tissue-engineered heart valves | |
| Mailand et al. | Surface and bulk stresses drive morphological changes in fibrous microtissues | |
| Tan et al. | Tissue engineering of retina and Bruch’s membrane: a review of cells, materials and processes | |
| IL296636A (en) | Collagen-based formulations usable as soft tissue fillers and/or implants | |
| Wang et al. | Application of bioprinting in ophthalmology | |
| Li et al. | Improved neuron culture using scaffolds made of three-dimensional PDMS micro-lattices | |
| Wang et al. | Digital micro-mirror device-based light curing technology and its biological applications | |
| Tsui et al. | Applications of three-dimensional printing in ophthalmology | |
| Baba et al. | Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft | |
| Danilevičius et al. | Direct laser fabrication of polymeric implants for cardiovascular surgery | |
| Lim et al. | Three-Dimensional Bioprinting of Biocompatible Photosensitive Polymers for Tissue Engineering Application | |
| Spoon et al. | Challenges of biological valve development | |
| Jonušauskas et al. | Femtosecond laser-made 3D micro-chainmail scaffolds towards regenerative medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20170829 |